1. Field of the Invention
This invention relates to methods for regulatable gene expression, detection and analysis of protein sequences, and detection and analysis of protein-protein interactions and agonists and antagonists thereof in multicellular organisms or cells therefrom.
This invention relates to interactions between biological molecules, particularly proteins, and methods for detecting and quantifying such interactions. The invention is particularly related to detection of protein-protein interactions that occur in the cellular cytoplasm of a cell from a multicellular organism. The invention further provides methods for detecting nuclear export sequences and nuclear localization sequences. This invention also relates to methods of regulatable gene expression. More specifically, the invention provides methods for inducible production of proteins in multicellular organisms or cells therefrom, including protein production from exogenously introduced GAL gene promoters, with or without galactose as the inducing molecule.
2. Background of the Related Art
The Human Genome Project has recently determined the complete human genetic sequence. From studies on only a very small fraction of the genome (likely less than 0.1%), it appears that protein-protein interactions most often comprise key mechanistic features of biological processes. Protein-protein interactions thus provide potential targets for therapeutic intervention in many disease states as well as manipulation of gene expression for any desired purpose.
Methods for detecting and analyzing of protein-protein interactions, as well as methods for achieving regulatable gene expression and detection and analysis of protein sequences, have been the focus of work based on several different native biological processes in a simple eucaryote, the yeast Saccharomyces cerevisiae. Such methods, which have generally employed native biological processes in S. cerevisiae, thereby using the yeast cell as the cellular platform, are recognized in the art as being useful in the biotechnology field, due in part to S. cerevisiae's genetic tractability and ease of biochemical and molecular manipulation. Thus, basic discoveries related to several native biological processes in S. cerevisiae have been exploited for detecting and analyzing protein-protein interactions and agonists and antagonists thereof, as well as methods of regulatable gene expression and detection and analysis of protein sequences in yeast cells.
An example of a system that has been exploited for detecting and analyzing protein-protein interactions and protein sequences and methods of regulatable gene expression in S. cerevisiae is the Gal3p-Gal80p-Gal4p gene switch, also known as the Y-GAL gene switch. The Y-GAL gene switch has been used in many biotechnology applications (U.S. Pat. No. 6,221,630 to Hopper; Mylin LM, et al., Methods Enzymol. 1990; 185:297–308; Cook J C, et al., Protein Expr Purif. December 1999; 17(3): 477–84; Ferreira B S, et al., Appl Microbiol Biotechnol. March 2003; 61(1): 69–76; Panuwatsuk W, et al., Biotechnol Bioeng. Mar. 20, 2003; 81(6): 712–8; Hwang W Z, et al., J Agric Food Chem. October 2001; 49(10): 4662–6; Brown R, et al., Enzyme Microb Technol. Jun. 1, 2000; 26 (9–10): 801–807; Yevenes A, et al., Biochimie. February 2000; 82(2): 123–7; Chen S, et al., Protein Expr Purif. December 1999; 17(3): 414–20; Hansen M K, et al., Receptors Channels. 1999; 6(4): 271–81; Hinz W, et al., FEBS Lett. Apr. 1, 1999; 448(1): 57–61; Hayden M S, et al., Protein Expr Purif. March 1998; 12(2): 173–84; Gerik K J, et al., J Biol Chem. Jan. 10, 1997; 272(2): 1256–62; Broder, E T, et al., Methods in Enzymology 1999 328: 430–443; Mitchell, D A, et al., 1993 Yeast 9: 715–723; Romanos, M A, et al., Yeast 8: 423–488). The Y-GAL gene switch is comprised of three proteins: i) a site-specific DNA binding transcriptional activator (Gal4p) that is tightly associated with its cognate DNA site (the UASGAL site) in the promoter regions of galactose-regulatable genes; ii) Gal80p, a protein that binds tightly to the transcriptional activation domain of Gal4p (amino acids 768–881) and inhibits Gal4p's capacity to recruit general transcription factors and RNA polymerase in the absence of galactose; and iii) Gal3p, a protein that binds to Gal80p in the presence of galactose and relieves Gal4p from Gal80p inhibition. Accordingly, Gal4p-mediated target gene expression occurs only in the presence of galactose (Johnston, M, 1987 Microbiol. Rev. 51:458–476).
The Y-GAL gene switch is a widely used tool for galactose-regulatable high level amplified expression of heterologous proteins in yeast (Mylin L M, et al., Methods Enzymol. 1990; 185:297–308; Cook J C, et al., Protein Expr Purif. December 1999; 17(3): 477–84; Ferreira B S, et al., Appl Microbiol Biotechnol. March 2003; 61(1): 69–76; Panuwatsuk W, et al., Biotechnol Bioeng. Mar. 20, 2003; 81(6): 712–8; Hwang W Z, et al., J Agric Food Chem. October 2001; 49(10): 4662–6; Brown R, et al., Enzyme Microb Technol. Jun. 1, 2000; 26 (9–10): 801–807; Yevenes A, et al., Biochimie. February 2000; 82(2): 123–7, Chen S, et al., Protein Expr Purif. December 1999; 17(3): 414–20; Hansen M K, et al., Receptors Channels. 1999; 6(4): 271–81; Hinz W, et al., FEBS Lett. Apr. 1, 1999; 448(1): 57–61; Hayden M S, et al., Protein Expr Purif. March 1998; 12(2): 173–84; Gerik K J, et al., J Biol Chem. Jan. 10, 1997; 272(2): 1256–62; Broder, ET, et al., Methods in Enzymology 1999 328: 430–443; Mitchell, D A, et al., 1993 Yeast 9: 715–723; Romanos, M A, et al., Yeast 8: 423–488). More recently, some of the instant inventors developed methodologies capable of detecting and characterizing protein-protein interactions, nuclear export and nuclear import sequences and galactose-independent inducibility of Gal4p-mediated gene expression in yeast by exploiting particular principles of the Y-GAL gene switch (see co-owned and co-pending U.S. patent application Ser. No. 10/165,873, incorporated herein in its entirety).
The protein-protein interaction method of Y- GAL gene switch methodologies is referred to as the “80-Trap method” and differs significantly from classical two-hybrid methods that depend on an intra-nuclear location of both interacting proteins. Specifically, in the 80-Trap method, the protein-protein interaction occurs within the cytoplasm but is reported (detected) by gene activation in the nucleus. Other methods provided in the Y-GAL gene switch methodologies are useful for detecting and analyzing nuclear export and nuclear import sequences. Additional methods provide a galactose-independent method of inducing Gal4p-mediated target gene expression. This last method provides for Gal4p-mediated activation of desired target genes by use of any low molecular weight, cell-permeant molecule that is a known effector of protein-protein interaction.
The methods provided, using the Y-GAL gene switch, exploit the fact that Gal4p's activation of target genes is regulated by Gal80p, a protein that binds to the Gal4p transcriptional activation domain and prevents it from recruiting general transcription factors required for recruiting RNA polymerase to the Gal4p-regulatable target promoter. These methods additionally take advantage of either one or more of three heretofore unappreciated mechanistic features of the Y-GAL gene switch. These newly-recognized mechanistic features are that i) Gal3p need not enter the nucleus to form a complex with the Gal80 protein in the presence of galactose; ii) when Gal80p, a nuclear/cytoplasmic shuttling protein, shuttles out of the nucleus it binds to cytoplasmically-restricted Gal3p; and, iii) binding of Gal80p to Gal3p in the cytoplasm reduces the amount of Gal80p in the nucleus available to bind to UASGAL-associated Gal4p at the gene promoter and, as a consequence, Gal4p-mediated transcriptional activation of Gal4p-regulated genes is increased. Thus, galactose-triggered interaction of Gal80p and Gal3p in the cytoplasm traps Gal80p in the cytoplasm and reduces the concentration of Gal80p in the nucleus. Consequently, the amount of Gal80p bound to Gal4p decreases, which results in Gal4p-mediated gene activation.
Unlike in yeast, there are very few existing methods for detecting and analyzing protein-protein interactions and agonists and antagonists thereof or regulatable gene expression in multicellular organisms or cells therefrom. Moreover, existing methods suffer from serious limitations. For example, one method to activate promoters in multicellular organisms or cells therefrom utilizes the Gal4 protein, the UASGAL-specific transcriptional activator of yeast referred to above. However, Gal4p expression in multicellular organisms or cells therefrom causes constitutive activation of target promoters containing the UASGAL site (Sadowski, I, Genetic Engineering, 1995 17:119–147; Horikoshi M, et al., Cell. Aug. 26, 1998; 54(5): 665–9; Webster N, et al., Cell. Jan. 29, 1998; 52(2): 169–78; Brand A H, et al., Development. June 1993; 118(2): 401–15; Scott K, et al., Cell. Mar. 9, 2001; 104(5): 661–73; Hrdlicka L, et al., Genesis. September–October 2002; 34(1–2): 51–7). A method to regulate Gal4p-mediated target gene expression in mammalian calls has been developed that takes advantage of the FLP recombinase target sequence (FRT) and recombination by the FLP recombinase. This method involves engineering into the same cells an FRT-associated GAL80 gene that can be excised by the FLP recombinase when the expression of the recombinase gene is induced by the heat shock promoter (Lee T, et al., Trends Neurosci. May 2001; 24(5): 251–4). This system has numerous significant problems, among them the heat shock method of induction and the extended persistence of the Gal80 protein, which can remain in the cells for as long as 48 hours after recombination excision of the GAL80 gene sequences.
Another existing method used in mammalian cells is the Gal4p-based two-hybrid system for analysis of protein-protein interactions (Sadowski I, Genetic Engineering, 1995 17: 119–147; Sadowski I, Anal Biochem. Feb. 15, 1998; 256(2): 245–7; Feng, Xin-Hua, et al., Two-Hybrid Systems 2001 177: 222–239. Humana Press, Edited by Paul N. MacDonald; Shioda T, et al., Proc Natl Acad Sci U S A. May 9, 2000; 97(10): 5220–4; Rojo-Niersbach E, et al., Biochem J. Jun. 15, 2000; 348 Pt 3: 585–90; Buchert M, et al., Biotechniques. September 1997; 23(3): 396–8, 400, 402; Luo Y, et al., Biotechniques. February 1997; 22(2): 350–2). One serious limitation of two-hybrid methods in multicellular organisms or cells therefrom is they are not applicable for a very large number of proteins including, but not limited to, most transcriptional activators, transcriptional repressors, RNA polymerase II components, components of the general (basal) transcription machinery, and proteins that are associated with chromatin or participate in chromatin remodeling. This limitation stems from the existing two-hybrid methods'detection of protein-protein interaction by activating reporter genes through either direct binding to the RNA polymerase or through binding to other proteins that in turn bind to an RNA polymerase subunit. Another serious limitation of existing methods is that they require that the relevant protein-protein interactions and the detection thereof take place in the nucleus at the reporter gene promoter. Thus, a protein that normally does not enter the nucleus is unlikely to participate in its normal protein interaction(s) if targeted to and sequestered in the nucleus. For example, post-translational modification (e.g., phosphorylation) of cytoplasmic proteins is often mediated by interaction with one or more cytoplasmically-confined proteins; this process is illustrated in many well-established membrane-based receptor signaling cascades wherein proteins are often tethered to membranes or in close proximity to membrane-associated proteins. Thus, direct targeting to the nucleus of a protein that normally would be post-translationally modified in the cytoplasm would prevent or severely hinder such modifications required for normal biological activity. Therefore, existing methods in the art requiring nuclear localization of both interacting proteins are not useful for detecting a significant fraction of protein-protein interactions that normally occur within the cell.
Given the limitations of existing analytical systems in multicellular organisms and cells therefrom, there is a need in the art for methods that provide a capacity to detect and analyze protein-protein interactions and agonists and antagonists thereof, detect and analyze protein sequences, and for regulatable gene expression in multicellular organisms or cells therefrom, including non-mammalian cells such as, for example, plant cells, particularly wherein such methods detect protein-protein interactions in the cytoplasm. Considering the highly regulated nature of most biologically-interesting genes studied to date and the fact that the estimated number of protein-coding sequences in the human genome will turn out to be only 25,000 to 30,000, it is important not to exclude a large fraction of genes from protein-protein interaction analyses, as occurs using the methods known in the art. In view of the large number of genetic sequences being determined, there is a need in the art for methods of identifying and characterizing the properties of the protein products encoded thereby in multicellular organisms (and cells thereof), including vertebrates, invertebrates and plants.